Just as the interaction between a pathogen and its host is an extremely complex dynamic, so too is the role of host cell death in immune defense and infection outcome. Not only can pathogen-induced host cell death occur by a variety of different mechanisms, but the consequences of cell death on immune response also vary with the different mechanisms. For example, cell death by apoptosis results in an inflammatory silent process in comparison to pyroptosis or necrosis and their associated inflammatory storms. A better understanding of this dynamic, the different cell death mechanisms, and their consequences will provide insight into new therapeutic approaches to control infection, at a time when such approaches are sorely needed in the face of rising antibiotic resistance and few new options on the horizon. Taking a chemical biological approach to studying pyroptotic cell death, we have identified two small molecules, 7-desacetoxy-6,7-dehydrogedunin (7DG) and pomiferin, which protect macrophages from pyroptotic cell death after exposure to anthrax lethal toxin. Using these probes, we are identifying new host factors and mechanisms involved in pyroptosis as well as mechanisms of cross-talk with other cell death pathways such as apoptosis. Specifically, by identifying the target of 7DG as protein kinase R (PKR), we have implicated a distinct role for PKR in pyroptosis that differs from its previously recognized role i apoptosis. Rather than serving as a kinase, PKR serves as a scaffold mediating protein-protein interactions critical for inflammasome activation. Among the proteins with which it interacts is a mitochondrial protein complex, thus contributing to the growing evidence for mitochondrial dysfunction in pyroptosis. Further, we have preliminary evidence that pomiferin also targets a mitochondria protein, cytochrome C oxidase, further converging on the mitochondria as a critical player in pyroptosis. In this proposal, we will work to characterize the mechanisms by which 7DG and pomiferin protect cells from pyroptotic cell death, including defining the roles of PKR and cytochrome C oxidase. This work will clarify the function of the mitochondria in pyroptosis and caspase-1 activation and help to elucidate the fine regulation that determines which cell death program is undertaken and what the outcome of infection will be. This understanding will inform the development of a new therapeutic paradigm of targeting the host in order to tip the balance of infection in favor of the host.